Summary While the Gag-5' leader interaction is crucial for the incorporation of HIV-1 genomes into virions, it is by no means the only RNA protein interaction that is required for the formation of morphologically accurate, infectious virions. Indeed, CRNA investigators have discovered non-canonical interactions between HIV-1 proteins (matrix, integrase, protease) and RNA molecules of host and/or viral origin that are, or may be, crucial for regulating the timing and location of key events in the formation of infectious virions. Additionally, CRNA investigators have made key advances in determining the structure of an RNA:RNA interaction (tRNA:U5-PBS) that is also initiated during virion morphogenesis and is similarly crucial for the generation of infectious virions. Project 4 will consist of a mixture of structural and functional experiments that will aim to elucidate the molecular details of these events and their importance during HIV-1 virion genesis. In Aim 1 Summers, Zhang and Bieniasz will determine the structures of matrix:tRNA complexes that appear to regulate Gag:membrane binding, in Aim 2 D?Souza, Smith, and Chiu will determine structures and functions of tRNA:U5-PBS:reverse transcriptase complexes that are formed during virion maturation, but are crucial for initiating subsequent reverse transcription. In Aim 3, Kutluay and Smith will determine the structure of IN:RNA complexes that appear crucial for the correct positioning of the ribonucleoprotein complex within the capsid core of mature virions, and will determine the feasibility of inhibiting this interaction with small molecules. Finally, in Aim 4 Swanstrom and Bieniasz will determine whether RNA:protease interactions that activate protease in vitro also occur in virions, as well as their functional significance, with a view to eventually determining the structural features of this interaction.